Liposome compositions comprising weak acid drugs and uses thereof

ABSTRACT

The present invention relates to a pharmaceutical composition comprising a weak acid drug, with the use of a bicarbonate salt to achieve a high incorporation of the drug into the liposome and a better therapeutic efficacy. Also disclosed is a method for treating a respiratory disease using the pharmaceutical composition disclosed herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Application No. 62/536,034,filed on 24 Jul., 2017 and U.S. Application No. 62/595,207, filed on 6Dec., 2017, the entire disclosures of which are incorporated herein byreference.

TECHNOLOGY FIELD

The present invention relates to a pharmaceutical composition with highdrug encapsulation efficiency or loading, methods of making and the usesof the pharmaceutical composition disclosed herein.

BACKGROUND OF THE INVENTION

Liposomes are vesicles formed by lipid bilayers containing an internalaqueous medium. Liposomes have been used as carriers for a variety oftherapeutic agents to offer improved delivery properties, such asenhanced blood circulation time, reduced cytotoxicity, sustained drugrelease, and specific drug delivery to selected tissues. When usingliposomes for therapeutic drug delivery, a higher drug encapsulationefficiency is desirable.

Currently, a number of drug-loading methods are available forincorporating drugs into liposomes. For weak acid drugs, liposomeshaving a higher inside/lower outside pH gradient has been reported fordrug loading, for example, in U.S. Pat. No. 5,939,096. The pH gradientis established by a salt of a weak acid, including carboxylic acid suchas formic acid, acetic acid, propanoic acid, butanoic acid, pentanoicacid, and substituted derivatives thereof. WO 96/25147 discloses the useof a bicarbonate salt for drug loading. However, later researchessuggest that the use of a bicarbonate salt in liposome preparationcauses gas accumulation that would disrupt and destabilize the liposomesand trigger premature drug release (Nahire et al. pH-triggeredechogenicity and contents release from liposomes, Mol Pharm. 2014 Nov.3; 11(11):4059-68; and Chen et al. A thermoresponsive bubble-generatingliposomal system for triggering Localized Extracellular Drug Delivery,ACS Nano. 2013 Jan. 22; 7(1):438-46), which suggest bicarbonate saltshould be avoided as a loading agent due to its disruptive anddestabilizing effect on the liposomes.

Many problems still remain unsolved in relation to weak acid drugs usingconventional liposomes, including poor drug loading and unsatisfactorycontrolled release rate. There is still an unmet need for liposomesuspensions with a high drug loading efficiency to increase therapeuticefficacy. The present invention addresses this need and other needs.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the present invention discloses a pharmaceuticalcomposition comprising one or more liposomes, comprising (a) a lipidbilayer, comprising at least one vesicle-forming lipid; and (b) aninternal aqueous medium inside the lipid bilayer comprising abicarbonate salt and a weak acid drug, wherein the liposomes aresuspended in an external medium and the molar ratio of the drug to thebicarbonate salt is from about 0.1:1 to 1:1.

Another embodiment of the present invention provides for apharmaceutical composition comprising one or more liposomes, comprising(a) a lipid bilayer, comprising at least one vesicle-forming lipid; and(b) an internal aqueous medium inside the lipid bilayer comprising abicarbonate salt and a weak acid drug, wherein the liposomes aresuspended in an external medium and the concentration of the bicarbonatesalt is about 50 mM to less than about 1000 mM.

A third embodiment of the present invention provides for apharmaceutical composition, comprising one or more liposomes, comprising(a) a lipid bilayer, comprising at least one vesicle-forming lipid; and(b) an internal aqueous medium inside the lipid bilayer comprising abicarbonate salt and a weak acid drug, wherein the liposomes aresuspended in an external medium and the pH of the external medium isabove the pK_(a) of the weak acid drug.

A fourth embodiment of the present invention provides for apharmaceutical composition, comprising one or more liposomes, comprising(a) a lipid bilayer, comprising at least one vesicle-forming lipid; and(b) an internal aqueous medium inside the lipid bilayer comprising abicarbonate salt and prostacyclin, wherein the liposomes are suspendedin an external medium and the molar ratio of the prostacyclin and thebicarbonate salt is about 0.1:1 to about 1:1.

A fifth embodiment of the present invention provides for apharmaceutical composition, comprising one or more liposomes, comprising(a) a lipid bilayer, comprising at least one vesicle-forming lipid; and(b) an internal aqueous medium inside the lipid bilayer comprising abicarbonate salt and prostacyclin, wherein the liposomes are suspendedin an external medium and the concentration of the bicarbonate salt isabout 50 mM to less than about 1000 mM.

In yet another embodiment, a pharmaceutical composition comprising oneor more liposomes, said liposome comprising (a) a lipid bilayer,comprising at least one vesicle-forming lipid; and (b) an internalaqueous medium inside the lipid bilayer comprising a bicarbonate saltand prostacyclin, wherein the pH of the external medium is above the pKaof prostacyclin is provided.

The present invention further provides a method for preparing thepharmaceutical composition disclosed herein, comprising the steps of:

(i) preparing a lipid solution using at least one vesicle-forming lipid;(ii) mixing the lipid solution from step (i) with a bicarbonate salt toform at least one liposome comprising a lipid bilayer and an internalaqueous medium, wherein the liposome is suspended in an external medium;(iii) adjusting the concentration of the bicarbonate salt in theexternal medium to produce a lower pH in the external medium of theliposome and a higher pH in the internal aqueous medium of the liposome,wherein the pH of the external medium is above the pK_(a) of a weak aciddrug; and(iv) adding the weak acid drug to the external medium.

Also provided are methods for treating a respiratory disease, comprisingthe steps of administering the pharmaceutical composition disclosedherein to a subject in need thereof.

The invention also provides the pharmaceutical composition disclosedherein for its use in the treatment and/or prophylactic treatment ofrespiratory disease.

The invention also includes the use of the pharmaceutical compositiondisclosed herein for the manufacture of a medicament for the therapeuticand/or prophylactic treatment of respiratory disease.

The details of one or more embodiments of the invention are set forth inthe description below. Other features or advantages of the presentinvention will be apparent from the following detailed description ofseveral embodiments, and also from the appending claims. Statementscontaining these terms should be understood not to limit the subjectmatter described herein or to limit the meaning or scope of the patentclaims below. This summary is a high-level overview of various aspectsof the invention and introduces some of the concepts that are furtherdescribed in the Detailed Description section below. This summary is notintended to identify key or essential features of the claimed subjectmatter, nor is it intended to be used in isolation to determine thescope of the claimed subject matter. The subject matter should beunderstood by reference to appropriate portions of the entirespecification, any or all drawings and each claim.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as is commonly understood by one of skill in theart to which this invention belongs.

As used herein, the articles “a” and “an” refer to one or more than one(i.e., at least one) of the grammatical object of the article. By way ofexample, “an element” means one element or more than one element.

The term “comprise” or “comprising” is generally used in the sense ofinclude/including which means permitting the presence of one or morefeatures, ingredients or components. The term “comprise” or “comprising”encompasses the term “consists” or “consisting of.”

All numbers are modified by the term “about”. As used herein, the term“about” refers to a range of ±10% of a specified value.

The term “subject” can refer to a vertebrate having a respiratorydisease or to a vertebrate deemed to be in need of treatment for arespiratory disease. Subjects include warm-blooded animals, such asmammals, such as a primate, and, more preferably, a human. Non-humanprimates are subjects as well. The term subject includes domesticatedanimals, such as cats, dogs, etc., livestock (for example, cattle,horses, pigs, sheep, goats, etc.) and laboratory animals (for example,mouse, rabbit, rat, gerbil, guinea pig, etc.). Thus, veterinary uses andmedical formulations are contemplated herein.

As used herein, “substantially free” means that pharmaceuticalcomposition contains less than 5%, 4%, 3%, 2% or 1% of a specificsubstance. In some embodiments, pharmaceutical composition does notcontain the specific substance.

The term “liposome” as used herein refers to microscopic vesicles orparticles made up of one or more lipid bilayers enclosing an internalaqueous medium. To form liposomes, the presence of at least one“vesicle-forming lipid” is needed, which is an amphipathic lipid capableof either forming or being incorporated into a lipid bilayer. Anysuitable vesicle-forming lipid may be used to form the lipid bilayerconstituting the liposomes. Vesicle-forming lipid includes, but notlimited to, phospholipids such as phosphatidylcholine (PC),phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid(PA), phosphatidyethanolamine (PE) or phosphatidylserine (PS), andcharged lipids, such as a positively charge lipid or a negativelycharged lipid.

The lipid bilayer of the liposome includes at least one vesicle-forminglipid and a sterol, which is selected from the group consisting ofcholesterol, cholesterol hexasuccinate, ergosterol, lanosterol, and anycombination thereof, but is not limited thereto. In an exemplaryembodiment, the sterol is cholesterol.

In some embodiments, the vesicle-forming lipid is a mixture of a firstphospholipid and a second phospholipid. In certain embodiments, thefirst phospholipid is phosphatidylcholine (PC), which is selected fromthe group consisting of hydrogenated egg phosphatidylcholine (HEPC),hydrogenated soy phosphatidylcholine (HSPC), dipalmitoylphosphatidylcholine (DPPC), distearyloyl phosphatidylcholine (DSPC),diarachidoyl phosphatidylcholine, dimyristoyl phosphatidylcholine(DMPC), egg phosphatidylcholine (EPC), soy phosphatidylcholine (SPC),oleoyl palmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine(DOPC), dipetroselinoyl phosphatidylcholine, palmitoylelaidoylphosphatidylcholine, palmitoyloleoyl phosphatidylcholine, dilauroylphosphatidylcholine (DLPC), diundecanoyl phosphatidylcholine, didecanoylphosphatidylcholine, dinonanoyl phosphatidylcholine, and any combinationthereof. In other embodiments, the second phospholipid is a polyethyleneglycol modified phospholipid, containing a polyethylene glycol having amolecular weight of about 500 to about 10,000 daltons, such as 1,2-distearoly-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG2000), a negatively charged phospholipid, such asdistearyloyl phosphatidylglycerol (DSPG),Dipalmitoylphosphatidylglycerol (DPPG) ordimyristoylphosphatidylglycerol (DMPG) or (DOPG). In an exemplaryembodiment, the mole percent of the first phospholipid:cholesterol:thesecond phospholipid is 50-70: 20-45:0.1-10, 50-70: 20-45:0.5-8 or55-65:25-40:1-6

In other embodiments, the vesicle-forming lipids is a mixture of a firstphospholipid and a charged lipid. In an exemplary embodiment,vesicle-forming lipids is a mixture of a first phospholipid, a secondphospholipid and a charged lipid. The charged lipid, includesstearylamine, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),3B-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol(DC-Cholesterol), N⁴—Cholesteryl-Spermine (GL67),dimethyldioctadecylammonium (DDAB),1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA),ethylphosphocholine (ethyl PC) or combination thereof. In anotherexemplary embodiment, the mole percent of the firstphospholipid:cholesterol:charged lipid is 50-70: 20-45:0.1-10, 50-70:20-45:0.5-8 or 55-65:25-40: 1-6.

In an embodiment, the mole % of HSPC, cholesterol, and DSPG in the lipidbilayer is 50-70: 20-45:0.1-10, 50-70: 20-45:0.1-5 or 55-65:25-40:0.5-8.In another embodiment, the mole % of HSPC, cholesterol and DSPE-PEG2000in the lipid bilayer is 50-70: 20-45:0.1-10, 50-70: 20-45:0.1-5 or55-65:25-40:0.5-8. In another embodiment, the mole % ofDPPC:Chol:DSPE-PEG2000 in the lipid bilayer is 50-70: 20-45:0.5-8,50-70: 20-45:0.1-5 or 55-65:25-40:1-6.

In one embodiment, the lipid bilayer of the liposomes may also includeat least one vesicle-forming lipid and a surfactant, which can be anon-ionic surfactant, a cationic surfactant or a zwitterionicsurfactant. A non-ionic surfactant has no formally charged groups in itshead. A cationic surfactant carries a net positive charge in its head. Azwitterion surfactant is electrically neutral but carries formalpositive and negative charges on different atoms.

Non limiting examples of non-ionic surfactant include non-ionic watersoluble mono-, di-, and tri-glycerides; non-ionic water soluble mono-and di-fatty acid esters of polyethyelene glycol; non-ionic watersoluble sorbitan fatty acid esters (e.g. sorbitan monooleates such asTWEEN 20 (polyoxyethylene 20 sorbitan monooleate), SPAN 80); non-ionicwater soluble triblock copolymers (e.g.,poly(ethyleneoxide)/poly-(propyleneoxide)/poly(ethyleneoxide) triblockcopolymers such as POLOXAMER 406 (PLURONIC F-127), or derivativesthereof.

Non-limiting examples of cationic surfactant includedimethyldialkylammonium bromide or dodecyltrimethylammonium bromide.

Non limiting examples of zwitterionic surfactant include 3-(N,N-dimethylpalmitylammonio)-propanesulfonate.

In some embodiments, the liposomes is substantially free of anionophore, which is a compound capable of facilitating the transport ofH⁺ or OH⁻ across the liposome membrane

A solvent for dissolving a vesicle-forming lipid for the preparation ofliposomes can be, for example, methanol, ethanol, ether, andcombinations thereof. Optionally, the solvent can be removed by asupercritical fluid later, and is preferably used in a minimum amount soas to decrease the time for performing an organic solvent removing step.

According to the present invention, the liposomes are prepared in amedium containing a bicarbonate salt. In particular, when thevesicle-forming lipid is in contact with a medium containing abicarbonate salt, a liposome suspension is formed.

A “bicarbonate salt” as used herein is preferably a pharmaceuticallyacceptable salt compound including a bicarbonate anion and a cationiccomponent. In one embodiment, the cationic component of the saltcompound is a metal. Non-limiting examples of the metal include a GroupIA or IIA metal, such as potassium (K), sodium (Na), calcium (Ca),magnesium (Mg), cesium (Cs), and lithium (Li) or a metal other thanGroup IA or IIA metal, such as ferrous iron (Fe) and nickel (Ni).Examples of bicarbonate salt include, but not limited to, potassiumbicarbonate, sodium bicarbonate, calcium bicarbonate, magnesiumbicarbonate, cesium bicarbonate, lithium bicarbonate, nickelbicarbonate, ferrous iron bicarbonate or any combination thereof.

The liposomes in the suspension is subjected to size reduction. Aliposome's size is typically referred to its diameter. Liposome sizereduction can be accomplished by a number of methods, such as extrusion,sonication, homogenization techniques or milling techniques, which arewell known and can be performed by persons skilled in this art.Extrusion includes passing liposomes, under pressure, one or more timesthrough filters having defined pore sizes. The filters are generallymade of polycarbonate, but can also be made of any durable materialwhich does not interact with the liposomes and which is sufficientlystrong to allow extrusion under sufficient pressure. The size of theliposomes can be reduced by sonication, which employs sonic energy todisrupt or shear liposomes that will spontaneously reform into smallerliposomes. For example, sonication can be conducted by immersing a glasstube containing the liposome suspension into the sonic epicenterproduced in a bath-type sonicator, or a probe type sonicator may be usedin which the sonic energy is generated by vibration of a titanium probein direct contact with the liposome suspension. In the presentinvention, the liposomes generally have a diameter of about 50 nm to 500nm, such as about 500 nm or less, about 400 nm or less, about 300 nm orless, about 200 nm or less or about 100 nm or less.

After sizing, the concentration of the bicarbonate salt in the externalmedium is adjusted to provide pH gradient between the internal aqueousmedium and the external medium, which can be carried out by a number ofways, for example, by exchanging the external medium with a suitablebuffer lacking bicarbonate salts, such as citric acid buffer (H₃C₆H₅O)and phosphoric acid buffer (H₃PO₄), by methods such as diafiltration,dialysis, ultrafiltration, or tangential flow filtration.

In one embodiment, the bicarbonate salt provides a lower outside and ahigher inside pH gradient between the external medium and the internalaqueous medium of the liposomes. In another embodiment, the pH of theinternal aqueous medium is at least one unit higher than the pH of theexternal medium. In yet another embodiment, the pH of the internalaqueous medium is about 7, 8, 9 or 10 and the pH of the external mediumis less than 7, less than 6, less than 5, less than 4, less than 3,about 3-7, about 3.5-6.5, or about 4-6. In yet another exemplaryembodiment, the pH of the external medium is above the pK_(a) of theweak acid drug.

The prepared liposome can be stored for substantial periods of timeprior to drug loading and administration to a subject. For example,liposomes can be stored at refrigerated conditions for substantialperiods of time prior to drug loading. Alternatively, liposomes can bedehydrated, stored, and subsequently rehydrated and loaded with one ormore active agents as needed, prior to administration. Liposomes mayalso be dehydrated after being loaded with one or more weak acid drug.Dehydration can be performed by a number of methods available and knownin the art. In some embodiments, liposomes are dehydrated using standardfreeze-drying apparatus i.e. dehydration under low pressure conditions.Also, liposomes can be frozen e.g. using liquid nitrogen. Saccharidescan be added to the liposomal environment, e.g., to the buffercontaining the liposomes, prior to dehydration, to ensure stability andintegrity of the liposome during dehydration. Examples of saccharidesinclude but are not limited to maltose, lactose, sucrose, trehalose,dextrose, sorbitol, mannitol, xylitol, or a combination thereof.

A liposome suspension having a lower outside/higher inside pH gradientas described above are ready for drug loading. Typically, a weak aciddrug to be loaded is added to the external medium of the liposome andthe resultant suspension is incubated under a condition to effect drugloading into the internal aqueous medium of the liposome, allowingdiffusion of the weak acid drug into the internal aqueous medium of theliposome and until a desired loading concentration and encapsulationefficiency (the percentage of the internal/encapsulated amount of thedrug relative to the total amount of the drug in the composition) isachieved.

A weak acid drug as used herein, unless indicated to the contrary orotherwise evident from the context, also include its pharmaceuticallyacceptable salt and its protonated form. In one embodiment, a weak aciddrug contains at least one functional group selected from the groupconsisting of a carboxyl group (—COOH), a hydroxyl group (—OH), aphosphate group (—PO₄) and any combination thereof. In anotherembodiment, a weak acid drug has a pKa of less than about 7, less thanabout 6, between 1 to less than about 7, between 2 to less than about 6,between 2 to 6.9, or between 2.5 to 6. A weak acid drug may also containone or more functional groups in addition to the above-mentionedcarboxyl group (—COOH), hydroxyl group (—OH), and phosphate group(—PO₄); such additional functional group(s) should not significantlychange the acidity of the drug from that of its non-functionalizedcounterparts. Table 1 shows the non-limiting examples of the weak aciddrug of the present invention.

TABLE 1 Characteristic of weak acid drugs suitable in the presentinvention Functional group Drug category Drug species pKa COOHProstaglandins Prostaglandin E1 (PGE1) 4.35 Prostaglandin E2 (PGE2) 4.3e.g., dinoprostone Epoprostenol 4.43 Iloprost 4.66 Beraprost 4.2MIRE-269 (ACT-333679) 3.77 Prostacycline 4.5 (treprostinil) Ralinepag(APD811) 3.5 Glucocorticoids Hydrocortisone sodium 3.66 (GCs) succinateMethylprednisolone 4.6 sodium succinate Methylprednisolone 4.29Hemisuccinate (MPSS) Salicylates Aspirin (acetylsalicylic 3.5 acid)Salicylic acid and other 2.97 salicylates Propionic acid Ibuprofensodium 5.2 derivatives Dexibuprofen 4.42 Naproxen sodium 4.15 Fenoprofen4.5 Ketoprofen sodium 4.76 Dexketoprofen 5.9 Flurbiprofen 4.3 Oxaprozin4.95 Loxoprofen 4.2 Acetic acid Indomethacin sodium 4.5 derivativesTolmetin 3.5 Etodolac 4.7 Ketorolac sodium 3.8 Diclofenac sodium 4.2Aceclofenac 4.7 Others Antibiotic 4.5 Cephalexin sodium Carbenoxolone4.7 sodium Chlorambucil 5.8 sodium OH Enolic acid Piroxicam 4.8 (Oxicam)Meloxicam 4.5 derivatives Lornoxicam 1.8 Warfarin 5.0 sodium PO₄Glucocorticoids Hydrocortisone sodium 1.18 (GCs) phosphate Betamethasonesodium 1.18 phosphate Dexamethasone sodium 1.89 phosphate Dexamethasone4.29 hemisuccinate COOH, OH Salsalate (Disalcid) 3.4

The present invention further provides a method for preparing thepharmaceutical composition disclosed herein, comprising the steps of:

(i) preparing a lipid solution using at least one vesicle-forming lipid;(ii) mixing the lipid solution from step (i) with a bicarbonate salt toform at least one liposome comprising a lipid bilayer and an internalaqueous medium, wherein the liposome is suspended in an external medium;(iii) adjusting the concentration of the bicarbonate salt in theexternal medium to produce a lower pH in the external medium of theliposome and a higher pH in the internal aqueous medium of the liposome,wherein the pH of the external medium is above the pK_(a) of a weak aciddrug; and(iv) adding the weak acid drug to the external medium.

In some embodiments, step (i) of the method comprises dissolving atleast one vesicle-forming lipid in an organic solvent to form a lipidsolution. The vesicle-forming lipid may be a mixture of a firstphospholipid and a second phospholipid or a mixture of a firstphospholipid and a charged lipid. In Step (ii), the lipid solution isthen mixed with an aqueous buffer solution of a bicarbonate salt of ametal to form a suspension which comprises an external medium and atleast one liposome, wherein the liposome is suspended in the externalmedium. In Step (iii), the concentration of the bicarbonate salt in theexternal medium is adjusted by replacing the external medium withanother buffer solution lacking bicarbonate salts to produce a loweroutside/higher inside pH gradient between the external medium and theinternal phase of the liposomes. In Step (iv), the weak acid drug isadded to the external medium of the suspension and the resultingsuspension is incubated under a predetermined condition to effect drugloading into the internal phase of the liposomes.

In some embodiments, the concentration of the bicarbonate salt in theinternal aqueous medium is about 50 mM to less than about 1000 mM inStep (iii) of the method.

In some embodiments, the pH of the external medium is above the pKa ofprostacyclin in step (iii) of the method. In other embodiments, the pHof the external medium is at least one unit lower than that of theinternal aqueous medium.

In some embodiments, the molar ratio of the drug to bicarbonate salt inthe internal aqueous medium is from about 0.1:1 to 1:1 in Step (iv) ofthe method.

Additionally, the present invention provides a method for utilizingfreeze-dried or frozen liposomes disclosed herein to load a weak aciddrug into the internal phase of the liposomes.

In some embodiments, the concentration of bicarbonate salt in in theinternal aqueous medium of the liposome is 50 mM or above, 100 mM orabove, 150 mM or above, 200 mM or above, 250 mM or above, 300 mM orabove, 350 mM or above, 400 mM or above, 450 mM or above, 500 mM orabove, 600 mM or above, 700 M or above, 800 mM or above and at most lessthan 1000 mM. In some embodiments, the concentration of bicarbonate saltin the aqueous buffer solution is from 50 mM to less than 1000 mM, from50 mM to 800 mM, from 200 mM to less than 1000 mM, from 200 mM to 800mM, or from 200 mM to 600 mM, from 250 mM to less than 1000 mM, from 250mM to 800 mM, or from 250 mM to 600 mM, from 300 mM to 600 mM. In anexemplary embodiment, the bicarbonate salt concentration in the internalaqueous medium of the liposome can be measured by any method known orwill be known in the art, or by the following steps: (a) separate thebicarbonate salt in the external medium from that of the internalaqueous medium by dialysis or size exclusion chromatography; (b) theliposome suspension without the bicarbonate salt in the external mediumis dissolved by methanol and the concentration of the bicarbonate saltin the internal aqueous medium is determined by ion chromatography. Insome embodiments, the external medium is substantially free ofbicarbonate salt.

In an embodiment, the pharmaceutical compositions of the presentinvention has a drug encapsulation efficiency of at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85% or at least 90%.

In another embodiment, the molar ratio of the drug to bicarbonate saltin the internal aqueous medium of the liposome (hereafter drug/saltmolar ratio) is about 0.1:1 to about 1:1. In an exemplary embodiments,the drug/salt ratio is from about 0.1:1 to 0.9:1, from about 0.1:1 to0.8:1, from about 0.1:1 to 0.7:1, from about 0.1:1 to 0.6:1 or fromabout 0.1:1 to 0.5:1.

In yet another embodiment, the weak acid drug encapsulated in theinternal phase of the liposomes is at a concentration from 1 mM to 800mM or above. In some embodiments, the drug encapsulated in the internalphase of the liposomes is at a concentration of 5 mM or above, 10 mM orabove, 20 mM or above, 40 mM or above, 50 mM or above, 80 mM or above,or 100 mM or above, and at most 800 mM.

According to the present invention, the pharmaceutical compositiondescribed herein has a higher drug encapsulation efficiency, anincreased molar ratio of the weak acid drug to the bicarbonate salt fromabout 0.1 to about 1, and a high encapsulated drug concentration.Without being bound by any particular theory, it is believed that higherencapsulated drug or drug loading in the pharmaceutical compositiondescribed herein delivers more drugs to the site of action, prolongs theeffect of the encapsulated drug and is more effective in reducing thesymptoms or signs of the disease.

The pharmaceutical composition of the present invention with a high drugencapsulation efficiency or a high drug loading as described herein canbe administrated at a therapeutically effective amount for treating arespiratory disease to a subject in need thereof. Examples ofrespiratory disease include, but are not limited to, pulmonaryhypertension, chronic obstructive pulmonary disease (COPD), asthma,cystic fibrosis, lower respiratory tract infection, bronchiectasis,bronchitis, bronchiolitis or croup. In an exemplary embodiment, therespiratory disease is pulmonary hypertension and the weak acid drug isprostacyclin or prostaglandin. In another exemplary embodiment, therespiratory disease is COPD and the weak acid drug is steroid orprostaglandin. In yet another exemplary embodiment, the respiratorydisease is asthma and the weak acid drug is steroid. In yet anotherexemplary embodiment, the respiratory disease is lower respiratory tractinfection and the weak acid drug is an antibiotic.

In general, the term “treating” as used herein refers to the applicationor administration of a pharmaceutical composition described hereinincluding at least one weak acid drug to a subject afflicted with arespiratory disease, a symptom or conditions of the respiratorydiseases, or a progression of the respiratory disease, with the purposeto cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve,or affect the disease, the symptoms or conditions of the disease, thedisabilities induced by the disease, or the progression of the disease.The term “therapeutically effective amount” used herein refers to theamount of the weak acid drug to confer a therapeutic effect in asubject. The therapeutically effective amount may change depending onvarious factors, such as administration route and frequency, body weightand age. Persons skilled in the art may determine the dosage in eachcase based on the disclosure herein, established methods, and their ownexperience.

In particular, the liposome composition comprising a weak acid drug asdescribed herein is administered by inhalation, injecting parenterally,i.e., intraarterialy, intravenously, intraperitoneally, subcutaneously,intra-vitreally, intrathecally, intraarticularly, intramuscularly,within other human body cavities, or dispersing via aerosol. Aerosoladministration methods include intranasal and pulmonary administration.In some embodiments, the liposome composition of the invention isadministered intravenously or intraperitoneally by a bolus injection orinfusion.

The present invention is further illustrated by the following examples,which are provided for the purpose of demonstration rather thanlimitation. Those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments which are disclosed and still obtain a like or similarresult without departing from the spirit and scope of the invention.

Example 1: The Effect of Different Salts for Drug Loading

1. Materials and Methods

1.1 PGE 1 Liposome Preparation Using Sodium Acetate (NaC2H₃O₂)

Liposome colloidal suspensions were prepared by ethanol injectiontechnique. Briefly, 496.8 mg of hydrogenated soy phosphatidylcholine(HSPC), 154.65 mg of cholesterol and 42.15 mg of1,2-distearoly-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG2000) were dissolved in 2.86 ml of ethanol. Thelipid solution in ethanol was injected into 17.4 ml of sodium acetate(200 mM or 400 mM) with vigorous stirring at 60° C. Liposomes wereformed as soon as the lipid solution was in contact with sodium acetate.This was followed by extruding the liposome suspension through a 200 nmand a 100 nm polycarbonate membrane (six times, respectively). Theliposome with about 100 nm was obtained and diafiltrated against acitrate buffer (pH 5.5) to form a higher inside/lower outside pHgradient between the external medium and the internal aqueous medium ofthe liposomes. The empty liposome suspension was then stored at 4° C.before drug loading process.

PGE1 (alprostadil) was added to the liposome suspension in the precedingparagraph at various drug/lipid molar ratios at room temperature (25°C.) for 20 min. The PGE1 loaded liposome suspension was stored at 4° C.

1.2 PGE 1 Liposome Preparation Using Sodium Bicarbonate (NaHCO₃)

The liposome suspension was prepared according to the method describedin Section 1.1, except that sodium acetate (200 mM or 400 mM) wasreplaced by sodium bicarbonate (e.g. 200 mM, 400 mM or otherconcentrations).

1.3 Quantitative Determination of Drugs

The amount of the drug encapsulated into the liposome was quantifiedusing Waters Alliance® HPLC system with a Photodiode Array (PDA)Detector. Briefly, an aliquot of 100 μL of each sample solution wasdirectly injected into the HPLC system with a mixture of methanol andphosphate buffer (6.7 mM conc., pH 3.0) at volume ratio of 80:20 as themobile phase at flow rate of 1.0 mL/min. Separation was performed in C18Column (Phenomenex Prodigy 5U ODS-3 150×4.6 mm), and then post-reactionwas carried out by mixed with 1N of KOH at 65° C. The peak of the druge.g. PGE1 was detected at 280 nm. The retention time was about 4.6minutes.

1.4 Encapsulation Efficiency

Encapsulation efficiency (EE), i.e. the percentage of the drugencapsulated (or loaded) in the internal aqueous medium of the liposomesto the total amount of the drug present in the liposome suspension (i.e.drug in the external medium and drug loaded into the internal aqueousmedium of the liposomes) was quantified as follows. Briefly, theliposome suspension was subjected to column chromatography. The free ornon-associated drug (i.e. the drug in the external medium) and theliposomes containing the encapsulated drugs in the internal aqueousmedium were separated from each other by eluting the liposome suspensionthrough a G-25 column. The amount of the free drug was determined usingWaters HPLC with PDA detection, and the amount of the encapsulated drugin the internal aqueous medium of the liposome, after destructing theliposomes with methanol (90% methanol and 10% liposome suspension), wasalso determined using the same method. The encapsulation efficiency wascalculated using the following formula,

${{{EE}(\%)} = {\left( \frac{L}{L + F} \right) \times 100}},$

where L is the amount of the encapsulated drug in the internal aqueousmedium of the liposomes, and F is the amount of the free drug in theexternal medium.

In addition, the internal drug concentration (mM) was calculated asfollows.

Internal drug concentration (mM)=[Drug [μg/mL]×EE %/molecularweight]/[trapped volume*]

(*In a specific condition, the trapped volume is 0.0314 mL per mL ofliposome suspension while the liposome particle size is around 110 nmand the phospholipid concentration is 10 mM. The trapped volume can becalculated by a method known in the art (Xu et al., InternationalJournal of Pharmaceutics (2012) 423(2), pp. 410-418.)

1.5 Mean Particle Size and Polydispersity Index (PdI)

The mean particle size of the liposomes was evaluated by dynamic lightscattering. The polydispersity index (PdI), a value indicating the sizedistribution of the liposomes, was determined using the same evaluationtechnique as for mean particle, with a particle size analyzer (BeckmanCoulter Delsa™ Nano C particle analyzer).

2. Results

2.1 Sodium Bicarbonate Offers a Higher Drug Encapsulation Efficiency

Table 2 shows the drug encapsulation efficiency (E.E.) and the drug tosalt molar ratio of the liposome suspensions using sodium bicarbonateand sodium acetate as the loading salts.

TABLE 2 The effect of bicarbonate salt and acetate salt for drug loadingMeasured Internal Salt drug Internal drug Molar ratio of Loadingconcentration concentration E.E. concentration internal drug SaltsFormulation [mM] (μg/mL) (%) (mM) to internal salt Sodium SA-200-1 20034.5 93.9% 2.91 0.015 Acetate SA-200-2 200 52.6 89.5% 4.23 0.021SA-200-3 200 209.8 85.6% 16.13 0.081 SA-400-1 400 26.5 90.6% 2.16 0.005SA-400-2 400 55.5 90.1% 4.49 0.011 SA-400-3 400 203.7 81.9% 14.99 0.037SA-400-4 400 435.4 66.6% 26.05 0.065 Sodium SB-200-1 200 31.9 97.9% 2.810.014 Bicarbonate SB-200-2 200 175.5 94.2% 14.85 0.074 SB-200-3 200642.7 95.1% 54.91 0.275 SB-200-4 200 934 95.9% 80.47 0.402 SB-200-5 2001860.8 86.7% 144.94 0.725 SB-200-6 200 2294 84.0% 173.12 0.866 SB-400-1400 18.7 97.3% 1.63 0.004 SB-400-2 400 56.53 97.0% 4.93 0.012 SB-400-3400 294 96.0% 25.36 0.063 SB-400-4 400 543 93.9% 45.81 0.115 SB-400-5400 1037.3 93.5% 87.14 0.218 SB-400-6 400 1940.8 92.8% 161.81 0.405SB-400-7 400 2727 73.3% 179.58 0.449 SB-400-8 400 2982.4 66.9% 179.250.448 a. The liposome composition includes 10 mM of phospholipid(HSPC:Chol:mPEG-DSPE = 3:1.91:0.072) b. Internal drug concentration (mM)= [Drug [μg/mL] × EE %/molecular weight]/[trapped volume (mL)*] *In theexample, the trapped volume is 0.0314 mL per mL of liposome suspensionwhile the particle size of liposome is around 110 nm and phospholipidconcentration is 10 mM.

The results in Table 2 indicate sodium bicarbonate increases the drugencapsulation efficiency of a weak acid drug and significantly increasesthe drug concentration within the internal aqueous medium of theliposomes, compared to sodium acetate. Specifically, the drug tobicarbonate salt molar ratio is less than 0.1:1 using sodium acetate asa loading agent and the molar ratio cannot increase any further bydoubling the concentration of sodium acetate, whereas the drug to saltmolar ratio is higher than 0.1:1 using sodium bicarbonate as a loadingagent.

Example 2: The Effect of Human Physiological Condition on thePharmaceutical Compositions Using Bicarbonate Salt for Drug Loading

Liposome suspensions using various concentrations of sodium bicarbonatefor PGE1 loading were prepared according to section 1.1 of Example 1.The liposome suspensions were incubated at 25° C. and 37° C. for 20minutes in an external buffer with osmolarity between 280-300 mOsm/Kg,to simulate human physiological condition.

TABLE 3 The effect of temperature on the pharmaceutical compositionswith various concentrations of sodium bicarbonate Bicarbonate Externalbuffer pH at different concentration Buffer Citrate incubationtemperatures (mM) (mOsm/Kg) (mM) pH 25° C. 37° C. 600 280 10 5.43 5.425.43 800 280 10 5.42 5.38 5.53 1000 280 10 5.78 5.37 6.68 (leakage) 1300280 10 5.5 5.44 6.74 (leakage)

Incubating formulations with a bicarbonate concentration of higher thanabout 1000 mM in human physiological condition (37° C. and 280-300mOsm/Kg) generated more CO2. and lead to liposome leakage. The leakedliposomes released bicarbonate salt and the drug from the internalaqueous medium into the external medium and increased the pH of externalmedium to higher than 6.5.

Example 3: The Effect of Different Bicarbonate Salt Concentrations onDrug Loading

The liposomal suspension was prepared according to the method in section1.1 of Example 1 and PGE1 was loaded with different concentrations ofsodium bicarbonate (50 mM, 150 mM, 200 mM, 250 mM, 300 mM, 400 mM, 500mM, 600 mM, and 800 mM).

Table 4 shows the effect of bicarbonate salt concentrations on drugloading.

TABLE 4 Internal Internal drug to Loading Internal [PGE1] [PGE1]internal salt Salts Salt [mM] (μg/mL) E.E. (%) (mM) Molar ratio Sodium 50* 226.4 93.8% 19.08 0.382 Bicarbonate 150* 700.7 95.7% 60.25 0.402200* 934.0 95.9% 80.47 0.402 250* 1446.4 92.3% 119.94 0.480 300* 1756.992.7% 146.32 0.488 400* 1940.8 92.8% 161.81 0.405  500** 1463.9 96.0%252.52 0.505 600* 2578.3 93.1% 215.66 0.359 800* 1614.9 90.2% 130.870.164 *10 mM of phospholipid **5 mM of phospholipid

The results suggest for a fixed amount of phospholipid, the drugencapsulation efficiency is above 90% and the drug to salt molar ratiois higher than 0.1:1 with 50 mM to 800 mM of bicarbonate salt.Bicarbonate salt concentration of 500 mM provided the highest drugencapsulation efficiency and drug to salt molar ratio.

Example 4: The Loading of Different Weak Acid Drugs Using BicarbonateSalt

A first (4-1) liposome suspension comprising HSPC, cholesterol andDSPE-mPEG2000 at a molar ratio of 3:2:0.075 was prepared according tothe method in Example 1.

A second (4-2) liposome suspension comprising HSPC, cholesterol and DSPGat a molar ratio of 3:2:0.075 was prepared according to the method inExample 1.

A third (4-3) liposome suspension comprising DPPC, Cholesterol andDSPE-mPEG2000 at a molar ratio of 3:2:0.075 was prepared according tothe method in Example 1.

A fourth (4-4) liposome suspension comprising DSPC, DPPC, Cholesteroland mPEG2000-DSPE at a molar ratio of 2.7:0.3:2:0.075 was preparedaccording to the method of Example 1.

A fifth (4-5) liposome suspension comprising HSPC, Cholesterol,mPEG2000-DSPE and stearylamine at a molar ratio of 3:2:0.075:0.025 wasprepared according to the method of Example 1.

A sixth (4-6) liposome suspension comprising HSPC, DMPC, Cholesterol,and mPEG2000-DSPE at a molar ratio of 2.7:0.3:2:0.075 was preparedaccording to the method of Example 1.

200 mM to 400 mM of bicarbonate salt was used to load various weak aciddrugs into the first, second and the third liposomal suspensions at 25°C. or 40° C. over 20 min. The loaded drug liposome suspensions werestored at 4° C.

Table 5 shows the bicarbonate salt is an effective loading agent forvarious weak acid drugs encapsulated in various liposomal formulations,with a high the drug encapsulation efficiency (higher than 80%) and drugto salt molar ratio greater than 0.1:1.

TABLE 5 The drug loading data of various weak acid drugs usingbicarbonate salt as a loading agent. Internal Measured Internal drug toInternal Salt Liposomal [drug] E.E. [drug] internal salt Weak Acid Drug[mM] Formulation (μg/mL) (%) (mM) molar ratio Treprostinil^(b) 400 (4-2)1530.0 97.6% 121.78 0.304 Treprostinil^(b) 400 (4-3) 1560 90.4% 115.010.288 Treprostinil^(b) 400 (4-5) 1500 93.7% 114.62 0.287Treprostinil^(b) 400 (4-6) 1550 94.5% 119.45 0.299 Treprostinil^(b) 250(4-4) 980 97.4% 77.84 0.311 MRE-269^(b) 400 (4-2) 174.6 98.7% 65.410.164 Iloprost^(b) 400 (4-2) 395.9 97.4% 170.34 0.426 PGE1^(a) 400 (4-1)1940.8 92.8% 161.81 0.405 Methylprednisolone 400 (4-1) 1132.2 85.9%65.27 0.163 Hemisuccinate (MPSS)^(b) Piroxicam^(b) 400 (4-1) 944.7 84.5%76.72 0.192 Meloxicam^(b) 400 (4-1) 1019.9 92.8% 85.78 0.214Lornoxicam^(b) 400 (4-1) 1106.3 93.3% 88.41 0.221 Ketorolac^(a) 400(4-1) 1192.7 91.2% 135.71 0.339 Ketorolac^(a) 200 (4-1) 766.8 91.0%87.06 0.435 Warfarin^(b) 400 (4-1) 1036.0 87.3% 93.42 0.234 ^(a)drugloading at 25° C., ^(b)drug loading at 40° C.

Example 5: The Effect of the pH of the External Medium of the LiposomeSuspension on Drug Loading

A first (5-1) liposome suspension comprising HSPC, cholesterol andmPEG2000-DSPE at a molar ratio of 3:2:0.075 and a second (5-2) and athird (5-3) liposome suspensions comprising HSPC, Cholesterol andmPEG2000-DSPE at a molar ratio of 3:2:0.28 were prepared. Treprostinilwas loaded into the liposome suspensions with 200 mM of bicarbonate.

The pH of the external medium of the first (5-1) liposome suspension isabove the pKa of the drug (the pKa of treprostinil is 4.5) and the pH ofthe external medium of the second liposome suspension (5-2) and thethird liposome suspension (5-3) are below the pKa of the drug.

Table 6 shows the pH of the external medium of the liposome suspensionplays an important role in drug loading. Specifically, the drugencapsulation efficiency is significantly higher if the pH of theexternal medium of the liposome suspension is above the pKa of the drugcompared to that of the liposome suspension that is below the pKa of thedrug (90.6% vs 27.3% and 33.5%). Similarly the molar ratio of internaldrug to salt is higher than 0.1:1 if the pH of the external medium ofthe liposome suspension is above the pKa of the drug compared to that ofthe liposome suspension that is below the pKa of the drug, which is lessthan 0.1:1.

TABLE 6 The effect of pH of the external medium of the liposomesuspension on drug loading Molar ratio of internal External MeasuredInternal drug to medium [drug] [drug] internal Formulation buffer/pH(μg/mL) E.E. (%) (mM) salt 5-1 10 mM citrate 300.0 90.6% 22.17 0.111buffer/pH 5.5 5-2 10% sucrose/ 315.9 27.3%* 7.03 0.035 pH 2.3 5-3 10%sucrose/ 353.1 33.5%* 9.65 0.048 pH 3.61 *some drug precipitation notedduring drug loading

Example 6: The Therapeutic Efficacy of Pharmaceutical Compositions withHigh Drug Loading

An in vivo evaluation of the therapeutic efficacy of the pharmaceuticalcomposition of Example 4 (the 4-2 liposome suspension encapsulated about1.5 mg of treprostinil using 400 mM bicarbonate salt) on pulmonaryhypertension was performed using 18 male Sprague Dawley rats, eachweighing approximately 300-350 g.

To induce pulmonary hypertension, a pressure catheter was inserted intothe pulmonary artery of each rat. One end of the catheter was exposed atthe nape of the neck and connected to a pressure transducer. 24 Hoursafter the catheter insertion, the rats were transferred into a hypoxicchamber where the oxygen (O₂) level was reduced to 10% (FiO₂=0.1) byincreasing the level of nitrogen (N₂). Once stable pulmonaryhypertension was established, each rat was administered with a singledose of the three test articles:saline (N=6), free treprostinil at 6μg/kg (N=6) or the pharmaceutical composition of Example 4 withtreprostinil at 6 μg/kg (liposomal treprostinil, N=6). The test articlewas administered using a microsprayer at the trachea bifurcation of therat. Following the test article administration, mean pulmonary arterialpressure (PAP) was measured at scheduled time points.

The results show the liposomal treprostinil is more effective inachieving a maximum reduction of mean PAP post injection compared tofree treprostinil (40% mean PAP reduction in the liposomal treprostinilgroup vs. 20% mean PAP reduction in the free treprostinil group).

What is claimed is:
 1. A pharmaceutical composition, comprising one ormore liposomes, said liposome comprising (a) a lipid bilayer, comprisingat least one vesicle-forming lipid; and (b) an internal aqueous mediuminside the lipid bilayer, comprising a bicarbonate salt and a weak aciddrug, wherein the liposomes are suspended in an external medium, and theconcentration of the bicarbonate salt is about 50 mM to less than about1000 mM.
 2. The pharmaceutical composition of claim 1, wherein the molarratio of the weak acid drug to the bicarbonate salt is from about 0.1:1to about 1:1.
 3. The pharmaceutical composition of claim 1, wherein thepH of the external medium is above the pK_(a) of the weak acid drug. 4.The pharmaceutical composition of claim 1, wherein the concentration ofthe bicarbonate salt is about 250 mM to about 800 mM.
 5. Thepharmaceutical composition of claim 1, wherein the bicarbonate saltprovides a pH gradient between the internal aqueous medium and theexternal medium and the pH of the internal aqueous medium is at leastone unit higher than the pH of the external medium.
 6. Thepharmaceutical composition of claim 1, wherein the encapsulationefficiency of the weak acid drug is at least 80%.
 7. The pharmaceuticalcomposition of claim 1, wherein the vesicle-forming lipid is a mixtureof a first phospholipid and a second phospholipid or a mixture of afirst phospholipid and a charged lipid.
 8. The pharmaceuticalcomposition of claim 7, wherein the first phospholipid is selected fromthe group consisting of phosphatidylcholine (PC), phosphatidylglycerol(PG), phosphatidylinositol (PI), phosphatidic acid (PA),phosphatidyethanolamine (PE), phosphatidylserine (PS) and anycombination thereof, the second phospholipid is a PEG modifiedphospholipid, a positively charged or a negatively charged phospholipid,and the charged lipid is a positively charged or a negatively chargedlipid.
 9. The pharmaceutical composition of claim 8, wherein the firstphospholipid is selected from HSPC, DSPC, DPPC, DMPC or combinationthereof and the second phospholipid selected from DSPG, DPPG, DMPG,PEG-DSPE, or combination thereof.
 10. The pharmaceutical composition ofclaim 8, wherein the first phospholipid is selected from HSPC, DSPC,DPPC, DMPC or combination thereof and the charged lipid is stearylamine,1,2-dioleoyl-3-trim ethyl ammonium-propane (DOTAP),3ß-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol(DC-Cholesterol), N⁴—Cholesteryl-Spermine (GL67),dimethyldioctadecylammonium (DDAB),1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA),ethylphosphocholine (ethyl PC) or combination thereof.
 11. Thepharmaceutical composition of claim 7, further comprising a sterolselected from the group consisting of cholesterol, cholesterolhexasuccinate, ergosterol, lanosterol, and combination thereof.
 12. Thepharmaceutical composition of claim 11, wherein the mole percent of thefirst phospholipid:cholesterol:the second phospholipid or the lipid is50-70: 20-45:0.1-10.
 13. The pharmaceutical composition of claim 1,wherein the bicarbonate salt is selected from the group consisting ofpotassium bicarbonate, sodium bicarbonate, calcium bicarbonate,magnesium bicarbonate, cesium bicarbonate, lithium bicarbonate, nickelbicarbonate, ferrous iron bicarbonate or combination thereof.
 14. Thepharmaceutical composition of claim 1, wherein the weak acid drug is aprostaglandin, a steroid, a non-steroidal anti-inflammatory drug(NSAID), or an anticoagulant.
 15. A method for treating a respiratorydisease, comprising the steps of administering a pharmaceuticalcomposition comprising one or more liposomes, said liposome comprising(a) an lipid bilayer, comprising at least one vesicle-forming lipid; and(b) an internal aqueous medium inside the lipid bilayer, comprising abicarbonate salt and a weak acid drug, wherein the liposomes aresuspended in an external aqueous medium, and the concentration of thebicarbonate salt is about 50 mM to about less than 1000 mM.
 16. Apharmaceutical composition, comprising one or more liposomes, saidliposome comprising (a) a lipid bilayer comprising at least onevesicle-forming lipid; and (b) an internal aqueous medium inside thelipid bilayer, comprising a bicarbonate salt and prostacyclin, whereinthe liposomes are suspended in an external aqueous medium, and theconcentration of the bicarbonate salt is about 50 mM to about less than1000 mM.